Shigellosis remains a major health problem worldwide. Shigella are unusual among intracellular bacterial enteropathogens in that they escape the phagocytic vacuole, and then spread within the host cell cytoplasm and pass into adjacent cells using the host cell cytoskeleton, thereby avoiding elements of the host defense system. Despite its central importance, little is known about the molecular basis of the interaction of the organism with the cytoskeleton. It derives from our previous work that the Shigella surface protein IcsA is essential to intracellular movement and cell-to-cell spread and that it interacts directly with the cytoskeleton in this process. Our long term objective is to understand the molecular mechanisms of both Shigella and macrophage determinants in this process and to examine the macrophage cytoskeletal signal transduction pathways utilized by Shigella. This research proposal specifically addresses the goal of the "characterization and modification of the molecules which regulate intracellular movement of microorganisms" within RFA AI-93-09. Our Specific Aims are as follows: (l) The localization of the Shigella surface protein IcsA to a single pole of the bacterium is both of central importance to Shigella movement using the host cell cytoskeleton and extremely unusual among bacterial surface proteins. We plan to investigate the molecular basis of the unipolar localization of IcsA. (2) We plan to characterize the mechanism of interaction of actin with IcsA. And, (3) we plan to identify and characterize additional bacterial factors involved in the interaction of Shigella with the cytoskeleton. Better understanding of the molecular mechanisms of the interaction of Shigella with the macrophage cytoskeleton will both enable more direct approaches to interrupting bacterial evasion of host defense and facilitate the development of therapeutics and vaccines that would target relevant molecules or signal transduction pathways.